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Introduction

293FT Cell Line

The 293FT Cell Line is a very suitable host for lentiviral production. The 293FT Cell Line is derived from the 293F Cell Line (see below) and stably expresses the SV40 large T antigen from the pCMVSPORT6TAg.neo plasmid. Expression of the SV40 large T antigen is controlled by the human cytomegalo-virus (CMV) promoter and is high-level and constitutive. For more information about pCMVSPORT6TAg.neo.

Use of the Cell Line

Studies have demonstrated maximal virus production in human 293 cells expressing SV40 large T antigen (Naldini et al., 1996), making the 293FT Cell Line a particularly suitable host for generating lentiviral constructs using the ViraPower™ Lentiviral Expression System (Catalog Nos. K4950-00 and K4960-00). Parental Cell Lines The 293 Cell Line is a permanent line established from primary embryonal human kidney transformed with sheared human adenovirus type 5 DNA (Graham et al., 1977; Harrison et al., 1977). The E1A adenovirus gene is expressed in these cells and participates in transactivation of some viral promoters, allowing these cells to produce very high levels of protein. The 293-F Cell Line (Catalog no. 11625) is a fastgrowing variant of the 293 cell line, and was originally obtained from Robert Horlick at Pharmacopeia.
Antibiotic Resistance

293FT cells stably express the neomycin resistance gene from pCMVSPORT6TAg.neo and should be maintained in medium containing Geneticin® at the concentration listed below. Expression of the neomycin resistance gene in 293FT cells is controlled by the SV40 enhancer/promoter.

The table below lists the recommended complete medium, freezing medium, and antibiotic concentration required to maintain and culture the 293FT Cell Line. Note: Fetal bovine serum should not be heat-inactivated for use with the 293FT Cell Line.

Complete Medium

[Antibiotic]

Freezing Medium

D-MEM (high glucose)

500 μg/ml Geneticin®

90% complete medium

10% fetal bovine serum (FBS)

10% DMSO

0.1 mM MEM Non-Essential Amino Acids (NEAA)

6 mM L-glutamine

1 mM MEM Sodium Pyruvate

1% Pen-Strep (optional)

D-MEM already contains 4 mM L-glutamine, which is enough to support cell growth of the 293FT Cell Line. However, since L-glutamine slowly decays over time, the complete medium needs to be supplemented with 2 m L-glutamine. This will ensure that the concentration of L-glutamine in complete medium will not get too low over time due to its slow degradation. Note: 293FT cells grow well in 6 mM L-glutamine, but higher concentrations of Lglutamine may reduce growth.

Use the following procedure to thaw 293FT cells to initiate cell culture. Thaw cells in prewarmed, complete medium without Geneticin®.

Remove the vial of frozen cells from liquid nitrogen and thaw quickly in a 37°C water bath.

Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol, and transfer the cells to a sterile 15 ml tube containing PBS. Briefly centrifuge the cells at 150-200 x g and resuspend the cells in 2 ml complete medium without Geneticin®.

Record the dilution factor. The dilution factor equals the total volume (amount of cell suspension and amount of trypan blue) divided by the amount of cell suspension.

Incubate the cells with the trypan blue solution for 1-2 minutes.

Count all cells (including the blue cells) using a Coulter Counter or manually using a hemocytometer chamber.

To calculate the total cells per ml in suspension, multiply the total count by the dilution factor.

To determine the viability, count only the blue cells. Calculate the % viability: [1.00 - (Number of blue cells ÷ Number of total cells)] x 100

Cell viability should be at least 95% for healthy log-phase cultures.

Subculturing Cells

Use this procedure to subculture 293FT cells grown in a T-75 cm2 flask. If you are using other-sized flasks, scale the reagent volumes accordingly.

Remove all medium from the flask and wash the cells once with 10 ml PBS to remove excess medium and serum. Serum contains inhibitors of trypsin.

Add 2 ml of trypsin/versene (EDTA) solution to the monolayer and incubate 1-5 minutes at room temperature until cells detach. Check the cells under a microscope and confirm that most of the cells have detached. If cells are still attached, incubate a little longer until most of the cells have detached.

Remove the cells from the tissue culture flask(s) following Steps 1-3, Subculturing Cells.

Determine viable and total cell counts and calculate the volume of freezing medium required to yield a final cell density of ≥ 3 x 106 cells/ml.

Prepare the required volume of freezing medium (see above).

Centrifuge the cells suspension (from Step 2) at 250 x g for 5 minutes in a table top centrifuge at room temperature. Carefully aspirate off the medium and resuspend the cell pellet in the pre-determined volume of chilled freezing medium.

Dispense aliquots of this suspension (frequently mixing to maintain a homogeneous cell suspension) into cryovials according to manufacturer’s specifications.

Freeze cells in an automated, controlled-rate freezing apparatus or using a manual method following standard procedures. For ideal cryopreservation, the freezing rate should be a decrease of 1°C per minute.

Transfer vials to liquid nitrogen storage.

Note: You may check the viability and recovery of frozen cells 24 hours after storing cryovials in liquid nitrogen by following the procedure outlined in Thawing Cells.

The 293FT Cell Line may be transiently transfected with any plasmid. General guidelines are provided below.

Make sure that cells are healthy at the time of plating. Overgrowth of cells prior to passaging can compromise their transfection efficiency.

On the day before transfection, plate cells such that they will be at the appropriate confluence at the time of transfection (see manufacturer’s recommendations for the transfection reagent you are using). Example: If you are using Lipofectamine® 2000 as a transfection reagent, plate cells such that they will be 90-95% confluent at the time of transfection.

Transfect your plasmid construct into the 293FT Cell Line using the method of choice (see above).

After transfection, add fresh growth medium containing 500 μg/ml Geneticin® and allow the cells to recover for 24-48 hours before proceeding to assay for expression of your gene of interest.

Generating Stable Cell Lines

293FT cells can be used as hosts to generate a stable cell line expressing your gene of interest from most plasmids (see Note below). Remember that the introduced plasmid must contain a selection marker other than neomycin resistance. Stable cell lines can then be generated by transfection and dual selection with Geneticin® and the appropriate selection agent.

Since 293FT cells stably express the SV40 large T antigen, we do not recommend generating stable cell lines with plasmids that contain the SV40 origin of replication.